An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS.

This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immuno-affinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures > 4 °C and pH values < 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (≥ ~3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.

Combining direct urinary injection with automated filtration and nanoflow LC-MS for the confirmatory analysis of doping-relevant small peptide hormones.

Nano-liquid chromatography (nanoLC) has proven itself as a powerful tool and its scope entails various applications in (bio)analytical fields. Operation at low (nL/min) flow rates in combination with reduced inner dimensions (ID < 100 µm), leads to significantly enhanced sensitivity when coupled with electrospray ionization-mass spectrometry (ESI-MS). Challenges that remain for the routine implementation of such miniaturized setups are related to clogging of the system and robustness in general, and thus the application of tedious sample preparation steps. To improve ruggedness, a filter placed upstream in the LC prevents particles from entering and clogging the system. This so-called online automatic filtration and filter back-flush (AFFL) system was combined with nanoLC and the direct injection principle for the sensitive confirmatory analysis of fifty different doping-relevant peptides in urine. The presented assay was fully validated for routine purposes according to selectivity and matrix interference, limit of identification (LOI), carryover, matrix effect, sample extract stability, analysis of educational external quality assessment (EQAS) samples, robustness of the online AFFL-setup and retention time stability. It was also fully compliant with the most recent minimum required performance levels (MRPL) and chromatographic/mass spectrometric identification criteria (IDCR), as imposed by the World Anti-Doping Agency (WADA). In the absence of labor-intensive sample preparation, the application of AFFL allowed for the injection of diluted urine samples without any noticeable pressure buildup in the nanoLC system. Contrary to earlier observations by our group and others, the addition of dimethylsulfoxide (DMSO) to the mobile phase did not enhance sensitivity in the presented nanoflow setup, yet was beneficial to reduce carry over. Although the robustness of the presented setup was evaluated only for the analysis of diluted urine samples, it is entirely conceivable that routine applications employing other matrices and currently running on analytical scale LC instruments could be transferred to micro/nanoLC scale systems to reach lower detection limits.

Doping control analysis of small peptides: A decade of progress.

Small peptides are handled in the field of sports drug testing analysis as a separate group doping substances. It is a diverse group, which includes but is not limited to growth hormone releasing-factors and gonadotropin-releasing hormone analogues. Significant progress has been achieved during the past decade in the doping control analysis of these peptides. In this article, achievements in the application of liquid chromatography-mass spectrometry-based methodologies are reviewed. To meet the augmenting demands for analyzing an increasing number of samples for the presence of an increasing number of prohibited small peptides, testing methods have been drastically simplified, whilst their performance level remained constant. High-resolution mass spectrometers have been installed in routine laboratories and became the preferred detection technique. The discovery and implementation of metabolites/catabolites in testing methods led to extended detection windows of some peptides, thus, contributed to more efficient testing in the anti-doping community.

A high-throughput assay for the quantification of intact Insulin-like Growth Factor I in human serum using online SPE-LC-HRMS.

Quantification of IGF-I is relevant in both doping control as a biomarker of growth hormone (GH) misuse in sports, and in the clinical field for longitudinal follow-up of patients with disorders related to the GH axis. Currently, better standardization of IGF-I measurements using mass spectrometry is in our best interest as it would enable long-term monitoring of an athletes' IGF-I levels by its addition to the Athlete Biological Passport (ABP). Here, a simplified and rapid top-down LC-HRMS method for quantification of IGF-I in human serum is presented. A ten-minute precipitation-based offline sample preparation is combined with online sample clean-up and separation on a conventional LC, resulting in a total runtime of nine minutes in between injections. The method was validated in the relevant range of 50-1000 ng/mL for the following parameters: linearity, precision, bias, Limit Of Quantification (LOQ), carry-over, selectivity, recovery and ion suppression. As proof of concept, the presented LC-HRMS assay was compared with results from a previous inter-laboratory study on intact IGF-I quantification using four human GH administration samples. It was additionally compared with the IDS-iSYS immunoassay using 47 athlete serum samples, showing good overall agreement with a slight positive bias of 24.2 ng/mL for the LC-HRMS assay at a mean sample concentration of 234 ng/mL. Also, a discrepancy between commercially available IGF-I reference material for the calibration of quantitative assays is discussed. This is of importance if LC-MS assays for IGF-I are to be harmonized.

Searching for new long-term urinary metabolites of metenolone and drostanolone using gas chromatography-mass spectrometry with a focus on non-hydrolysed sulfates.

Sulfated metabolites have been shown to have potential as long-term markers of anabolic-androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography-mass spectrometry (GC-MS) with non-hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC-MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long-term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC-chemical ionization-triple quadrupole MS (GC-CI-MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli-derived ß-glucuronidase, Helix pomatia-derived ß-glucuronidase/arylsulfatase and non-hydrolysed sulfated steroids). For metenolone, a non-hydrolysed sulfated metabolite, 1ß-methyl-5α-androstan-17-one-3ζ-sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non-hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α-hydroxy-2α-methyl-5α-androstan-17-one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).

Urinary detection of rapid-acting insulin analogs in healthy humans.

Human insulin and its synthetic analogs are considered as life-saving drugs for people suffering from diabetes mellitus. Next to the therapeutic use, scientific and non-scientific literature (e.g. bodybuilding forums; antidoping intelligence and investigation reports) indicate that these prohibited substances are used as performance enhancing agents. In the present report, the development and validation of a sensitive analytical strategy is described for the urinary detection of three rapid-acting insulin analogs (Lispro, Aspart, Glulisine). The method is based on sample purification by the combination of ultrafiltration and immunoaffinity purification and subsequent analysis by nano-flow liquid chromatography coupled to high resolution mass spectrometry. Next to the results on different validation parameters (LOD: 10 pg/mL; recovery: 25-48%; matrix effect: -3-(-8) %), data on urinary elimination times, which were obtained in the frame of an administration study with the participation of healthy volunteers, are presented. The determined detection windows (~9 hours) are expected to help to evaluate current routine analytical methods and aim to aid doping authorities to set appropriate target windows for efficient testing.

Gas chromatography-mass spectrometry analysis of non-hydrolyzed sulfated steroids by degradation product formation.

Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non-hydrolyzed sulfated steroids with gas chromatography-mass spectrometry (GC-MS) instead of the conventionally applied liquid chromatography-mass spectrometry (LC-MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization-GC-triple quadrupole MS (CI-GC-TQMS) and low energy-electron ionization-GC-quadrupole time-of-flight-MS (LE-EI-GC-QTOF-MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC-MS, forming two isomers. In CI, the dominant species (ie, [MH - H2 SO4 ]+ or [MH - H4 S2 O8 ]+ for bis-sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE-EI, [M - H2 SO4 ].+ and/or [M - H2 SO4 - CH3 ].+ are the dominant species in most cases. Based on the common GC-MS behavior of non-hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC-MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC-MS and LC-MS analysis of non-hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC-MS after hydrolysis. For non-hydrolyzed sulfated steroids, GC-MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC-MS.